Benefits of peptide drugs include lower toxicity and greater efficacy than existing drugs and in some cases, new classes of compounds that are otherwise unavailable. However, the development of a peptide lead to a commercial drug continues to be a very challenging and time consuming process. Peptides generally suffer from poor in vivo stability, poor pharmacokinetics, and poor bioavailability. In search of better peptide scaffolds for drug development, researchers have recently focused on highly constrained peptides known as cyclotides as extremely stable and versatile scaffolds for the production of high affinity ligands. Importantly, cyclotide scaffolds have demonstrated a wide spectrum of biological activities and in some cases oral bioavailability. Other cyclotides have been shown to cross the cell membrane through macropinocytosis. Finally, cyclotide structures can be encoded and expressed in bacteria or animal cells, and are amenable to substantial sequence variation. Thus, we believe that cyclotide drugs could provide the opportunity to greatly expand the number of "druggable" targets by targeting the thousands of intracellular protein-protein interactions that cannot be functionally modulated by current therapeutics. We propose to use fluorescent proteins as a FRET-couple to screen genetically-encoded libraries of cyclotides inside living bacterial cells for potential inhibitors of NF?B signaling and inflammatory responses. NF?B is a transcription factor with an important role in regulating immune and inflammatory responses. It mediates the biological actions of TNFa and abnormalities in TNFa/NF?B signaling play critical roles in inflammatory diseases such as arthritis, atherosclerosis and Crohn's disease. As a consequence, drugs blocking TNFa/NF?B signaling could have a number of therapeutic actions. A key factor controlling the actions of NF?B and mediating the effects of TNFa is I?B kinase (IKK). IKK activity is modulated by an allosteric activator, NEMO. Thus, cyclotide inhibitors of the NEMO/IKK[unreadable] interaction will likely block TNFa stimulation of NF?B signaling and might be useful as anti-inflammatory drugs. Discovery of selective cyclotide inhibitors of NEMO/IKK[unreadable] binding would be facilitated (as proposed here) through the power of molecular evolution strategies and high throughput cell based screening using fluorescence-activated cell sorting (FACS) to enable generation and selection of compounds with optimal binding and inhibitory characteristics. PUBLIC HEALTH RELEVANCE: The concept of using peptides to modulate intracellular processes has been investigated for decades, as peptides play a central role in every cell in the body. These strategies have historically failed because most peptides lack the ability to enter cells, and linear peptides are inherently unstable within the body. We propose development of a breakthrough class of peptide drugs called cyclotides that are rather stable and biologically active, with good drug-like properties, including resistance to proteolytic degradation and the ability to cross mammalian cell membranes. This class of compounds represents a solution for modulating intracellular protein-protein interactions, which have been identified as critical control points for most human diseases. Specifically, we propose to deploy the power of molecular evolution strategies and high throughput cell based screening to enable generation and selection of cyclotides that could inhibit NF?B signaling. Abnormalities in TNFa/NF?B signaling play critical roles in inflammatory diseases such as arthritis, atherosclerosis and Crohn's disease. As a consequence, drugs blocking TNFa/NF?B signaling could have a number of therapeutic actions as anti-inflammatory agents. Specifically, small molecule drugs that block the binding interaction of NEMO to IKK[unreadable] inhibit NF?B signaling could be developed as anti-inflammatory drugs.